十字花科中硫甙多样性及代谢.pdf

Glucosinolate biochemical diversity and innovation in the Brassicales Richard Mithen a Richard Bennetta Julietta Marquezb aInstitute of Food Research Colney Lane Norwich NR4 7UA UK bDivision of Agricultural and Environmental Sciences University of Nottingham Sutton Bonington LE12 5RD UK a r t i c l ei n f o Article history Received 30 July 2010 Received in revised form 24 September 2010 Keywords Brassicales Capparaceae Forchhammeria Setchellanthus Glucosinolates Herbarium specimens a b s t r a c t Glucosinolates were analysed from herbarium specimens and living tissues from representative of all families of the Brassicales following the phylogenetic schemes of Rodman et al 1998 and Hall et al 2002 2004 including specimens of Akania Setchellanthus Emblingia Stixis Forchhammeria and mem bers of the Capparaceae for which glucosinolate content had not previously been reported The results are reviewed along with additional published data on glucosinolate content of members of the Brassi cales In addition to providing an overview of the evolution of glucosinolate biochemical diversity within the core Brassicales there were three main fi ndings Firstly the glucosinolate content of some orphan taxa of the Brassicales such as Setchellanthus and Emblingia were consistent with recent phylogentic anal yses based upon DNA sequence comparisons while further analyses of Tirania and Stixis is required Sec ondly methyl glucosinolate is found within the Capparaceae and Cleomaceae but also unexpectedly within Forchhammeria with implications for the biochemical and evolutionary origin of methyl glucosin olate and the phylogenetic relationships of Forchhammeria Thirdly whereas Old World Capparaceae con tain methyl glucosinolate New World Capparaceae including New World Capparis either contain methyl glucosinolates or glucosinolates of complex and unresolved structures indicative of continued innovation in glucosinolate biosynthesis These taxa may be productive sources of glucosinolate biosynthetic genes and alleles that are not found in the model plant Arabidopsis thaliana 2010 Elsevier Ltd All rights reserved 1 Introduction PlantswithintheBrassicalesaccumulateb thioglucoside N hydroxysulfates commonly known as glucosinolates Fig 1 In recent years there have been major advances in our understanding of the molecular genetics and biochemistry of glucosinolate bio synthesis and accumulation Glucosinolates are synthesized from a small number of primary amino acids Leu Ile Val BCAA Met Phe or Tyr and Trp through three stages Sonderby et al 2010 Firstly the amino acids may be elongated by the insertion of methylene groups de Quiros et al 2000 This is a multistep cyclic process which may involve multiple up to 10 methylene insertions Secondly the primary or elongated amino acids are metabolised to provide the core glucosinolate structure This in volves an initial conversion to an aldoxime via the activity of cyto chrome p450s of the CYP79 family that are specifi c to the amino acid precursor Chen et al 2003 Mikkelsen and Halkier 2003 Reintanz et al 2001 The aldoxime is subsequently converted to an activated compound via a CYP83 enzyme Bak and Feyereisen 2001 Naur et al 2003 which is then conjugated with glutathione to produce S alkyl thiohydroximates Geu Flores et al 2009 which are converted to thiohydroximates by the C S lyase SUR1 Mikkelsen et al 2004 These highly reactive intermediates are glycosylated to form desulphoglucosinolates and sulphated to pro duce glucosinolates Thirdly the side chain derived from the amino acid precursor can be decorated in a variety of ways including hydoxylation alkenylations and methoxylations Parkin et al 1994 The interaction between the initial elongation of the amino acid precursor with subsequent side chain modifi cations post syn thesis results in the 120 or so glucosinolates that have so far been described The conversion of BCAA and Phe to an aldoxime is also common to the synthesis of cyanogenic glycosides that are wide spread in the pant kingdom Rodman et al 1998 It is likely that the evolutionary origin of glucosinolate biosynthesis in the Brassi cales is associated with diversion of the aldoxime away from cya nogenic glycosides biosynthesis through the activity of a mutated CYP83 like enzyme and subsequent detoxifi cation of the reactive product through sulphation and glycosylation to produce glucosin olates Halkier and Gershenzon 2006 The signifi cant advances in our understanding of glucosinolate biosynthesis have arisen largely through the use of Arabidopsis tha liana and as a consequence have focused almost exclusively on Met derived and Trp derived glucosinolates that accumulate in this species Thus aspects of the use of BCAA and Phe particularly 0031 9422 see front matter 2010 Elsevier Ltd All rights reserved doi 10 1016 j phytochem 2010 09 017 Corresponding author Current address CITAB UTAD Centre for the Research and Technology for Agro Environment and Biological Sciences Universidade de Tras os Montes e Alto Douro Apartado Portugal Tel 44 1603 255259 fax 44 1603 507723 E mail address Richard mithen bbsrc ac uk R Mithen Phytochemistry 71 2010 2074 2086 Contents lists available at ScienceDirect Phytochemistry journal homepage 十字花目 硫甙的合成过程 their elongated forms as precursor amino acids required further exploration Moreover the biosynthesis of methyl glucosinolate which is the principle glucosinolate accumulating in many species of the Capparaceae and Cleomaceae has not been explored This glucosinolate may be derived from alanine as a novel glucosinolate precursor possibly through the action of a further CYP79 or it may arise as a result of the single or multiple cleavage of side chains de rived from Met or BCAA or elongated Phe Concurrently with increasing knowledge on the molecular genetics of glucosinloates there have been major advances in our understanding of the evolutionary relationships of taxa within the Brassicales through the use of DNA sequence comparisons The current view of this order is similar to the radical suggestion proposed by Dalhgren 1975 in which several taxa of diverse hab its and morphologies were grouped together within the Brassi cales partly as they all produced glucosinolates Rodman et al 1998 used sequence comparisons of the chloroplast rbcL gene and nuclear 18S ribosomal RNA gene to provide an overview of the entire order Fig 2 but with limited analyses of the relatively large Brassicaceae and Capparaceae sensu lato families Rodman et al 1998 In this analysis Setchellanthus caeruleus considered at the time to be within the Capparaceae was not clustered with other members of Capparaceae but was placed within the basal Brassicaleantaxa Subsequentstudiesonmorphology Iltis 1999 reproductive anatomy Tobe et al 1999 palynology Tomb 1999 and further molecular sequence analyses Karol et al 1999 led to S caeruleus being placed within a new monospecifi c family Setchellanthaceae allied to the basal families of the Brassicales Iltis 1999 Fig 2 In addition in a somewhat analogous manner to Setchallanthus DNA analyses also confi rmed the assignment of the western Australian shrub Emblingia calceolifl ora the only member of the family Emblingaceae to the Brassicales Chandler and Bayer 2000 Hall et al 2004 The two largest families within the Brassicales are the Brassic aceae approximately 350 genera and 3000 species and Cappara ceae sensu lato approximately 45 genera and 800 species with the later having two major subfamilies the Cleomoideae and the Capparoideae It has been clear for some time that while the Brass icaceae are morphologically distinct from the Capparaceae due to the cruciform corolla tetradynamous stamens and characteristic silique fruit these families subfamilies are on the basis of molec ular sequence analyses monophylic with the Cleomoideae being more closely related to the Brassicaceae than to the Capparoideae Hall et al 2002 reported more extensive molecular analyses of the Capparaceae and Brassicaceae than that of Rodman et al 1998 This study provided considerable support for the thesis that there are three major clades within these two families and support the recognition of three monophyletic families the Cap paraceae sensu stricto Cleomaceae and Brassicaceae APG 1998 with the latter two diverging from each other after divergence from the lineage leading to the Capparaceae sensu stricto Hall et al 2002 This familial classifi cation is used for the rest of this paper In addition various important subdivisions within the Cleoma ceae and Capparaceae were identifi ed and Capparis has been shown to be diphyletic In the Capparaceae there is a New World clade comprising for example New World Capparis Morisonia Steriphoma and Atamisquea and a Old World clade comprising gen era such as Old World Capparis Maerua Boscia and Thylachium in addition to a third clade containing the pantropical genus Crateva Within Cleomaceae there are two distinct clades one comprising the temperate New World taxa such as Oxstylis Wislizenia Cleom ella and Isomeris and the remaining clade containing all other taxa Hall et al 2002 The four genera Forchhammeria Tirania Stixis and Neothorelia conventionally placed within the tribe Stixae have recently been excluded from the Capparaceae on the basis of fl oral characteristics Kers 2003 Following DNA sequence analyses Forchhammeria and Tirania have been associated with the Resedaceae and Gyroste monaceae Hall et al 2004 2002 referred to as the GRFT clade Fig 3 O S HO OH OH OH NOSO3 R Fig 1 Glucosinolate structure The side chain R is derived from amino acids Capparis Cleome Arabidopsis Brassica Reseda Gyrostemnon Tovaria Pentadiplandra Koerberlinia Batis Salvadora Setchellanthus Limnanthus Floerka Carica Moringa Tropaeolum Bretschenidera Akania Capparaceae Cleomaceae Brassicaceae Resedaceae Gyrostemonaceae Tovariaceae Pentadiplandraceae Koeberliniaceae Bataceae Salvadoraceae Setchellanthaceae Limnanthaceae Caricaceae Moringaceae Tropaeolaceae Akaniaceae Aromatic and BCAA GSLs Indole GLs Chain elongated GSLs Methionine derived GSLs Methyl GSLs Undetermined GSLs CoreBasal Fig 2 Phylogeny of the Brassicales inferred from combined rbcL and 18S nuclear DNA sequence analyses from Rodman et al 1998 showing the cumulative increase in biochemical diversity Glucosinolates do not occur in Koerberliniaceae Indole glucosinolates can be detected at trace amounts in basal families R Mithen et al Phytochemistry 71 2010 2074 20862075 分子比对 进化 In this paper we analyse glucosinolates extracted from seed or herbarium tissue from representative of all the families within the Brassicales but with an emphasis on taxa that have not been ana lysed previously including Akania Setchellanthus Forchhammeria Emblingia and several taxa in the Gyrostemonaceae Capparaceae and Cleomaceae and then comment on relationship between biochemical structure and phylogeny Of particular interest is the distribution of methyl glucosinolates 2 Results and discussion Glucosinolates were successfully extracted and analysed from 156 of 189herbarium specimens Table 1 The absence of glucosin olates from 33 specimens could either have arisen from a lack of glucosinolate biosynthesis which is likely to be the case with Koeb erlinia or due to glucosinolate loss and degradation associated with tissue disruption following collection and drying This is likely to be the case with taxa such as the halophytic Batis and those from the wet tropics such a Tirania and Stixis The age of the specimen was not a factor with glucosinolates extracted from Akania and Brets chiedera collected in 1917 and 1919 respectively being of similar abundance to those from for example Setchellanthus collected in 1986 Fig 4 Only trace amounts of glucosinolates were detected inaspecimenofE calceolifl oracollectedin1850 butthiswaslargely due to the extremely small amount of tissue available The amount of tissue available from the majority of herbarium specimens en abled analyses of both desulpho and native glucosinolates by LC MS but not NMR analyses that would be required for resolution of isomeric structures of glucosinolates with identical mass Thus the mass of glucosinolates together with retention times and previ ous studies enabled reasonable assumptions of chemical composi tion to be made but without knowledge of isomeric structures particularly with regard the precise position of hydroxyl and meth oxy groups and the extent of aliphatic side chain branching In gen eral different specimens of the same species gave similar results 2 1 Akaniaceae Stapf including Bretschenideraceae Engl and Gilg Glucosinolates were extracted from leaves of three herbarium specimens of Akania bidwillii endemic to eastern Australia Two of the specimens had been collected from the wild in the year 1917 and 1966 respectively while the third was from a tree culti vated in the UK and collected in 1976 Table 1 All tissue contained benzyl hydroxybenzyl dihydroxybenzyl and methoxybenzyl gluc osinolates As far as we are aware there are no other reports of glucosinolates in Akania Trace amounts of 4 methoxyindolymeth yl glucosinolate can be detected in the extracts from Akania but these are only apparent via detection of specifi c characteristic ions by mass spectrometry the levels being below the threshold for UV detection Tissue was analysed from leaves of a single specimen of Bretschenidera sinensis collected from China in 1919 Hydroxy methylpropyl benzylandhydroxybenzylglucosinolatewere Capparis hastata Maerua kirkii Apophyllum anomalum Crateva palmeri Pondandrogyne chiriquensis Cleome pilosa Cleome viridiflora Polanisia dodecandra Wislizenia refracta Arabidopsis thaliana Capsella spp Nasturtium officinale Stanleya pinnata Aethionema saxatile Aethionema grandiflora Tirania purpurea Forchhammeria pallida Forchhammeria watsonii Forchhammeria sp nov Forchhammeria sessifolia Forchhammeria trifoliata Reseda lutea Tersonia cynthiflora Oligomeris Tovaria pendula Gyrostemon sp Pentadiplandra brazzeana Emblingia calceoliflora Koeberlinia spinosa Batis maritima Carica papaya Moringa oleifera Tropaeolum majus Gyrostemon tepperi Bretschneidera sinesis 0 01 substitutions site Capparaceae Methyl GSL Brassicaceae Forchhammeria Resedaceae Gyrostemonaceae Methyl GSL Chain elongated Met derived GSLs Undescribed GSLs Cleomaceae Chain elongated Met derived GSLs TiraniaNo detectable GSLs Methyl GSL Basal Chain elongated Phe derived GSLs Chain elongated BCAA GSLs Core Phe derived GSLs BCAA derived GSLs Trp derived GSLs GRFT clade Fig 3 Phylogeny of core Brassicales showing the GRFT clade inferred from combined rbcL ndhF and matK sequence analyses from Hall et al 2004 aligned with glucosinolate profi les Glucosinolates were analysed from representative species from all these major divisions see Tables 1 and 2 and text but not necessarily in the species used for the phylogenetic analyses Glucosinolates were not found in Koeberlinia and Tirania 2076R Mithen et al Phytochemistry 71 2010 2074 2086 Table 1 Herbarium specimens taxonomic names are as they appear on herbarium sheets from which glucosinolates were extracted TaxonCollectorHerbariumDate of collectionCountry of collection Akaniaceae Akania hillii Hook fLongman HAKew1917Australia Akania lucens F Muell Airy Shaw000 69 15858Kew1976cultivated UK Akania lucens F Muell Airy ShawHayes HC et al 2507Kew1966Australia NB These species are synonyms of Akania bidwillii R Hogg Mabb Bretschneideraceae Bretschiedera sinensis HemsleyWang Te Hui 12 130Kew1919China Setchellanthaceae Setchellanthus caeruleus BrandegeeNPT 246Kew1986Mexico Setchellanthus caeruleus BrandegeeIltis and Lasseipue 137WiscMexico Salvadoraceae Dobera loranthifolia Warb Harms Magogo FC Innes RR RRI 355Kew1978Tanzania Dobera loranthifolia Warb HarmsHooper SS Townsend CC 1184Kew1977Kenya Dobera glabra Forsk Poir Carter S Stannard B 142Kew1977Kenya Azima tetracantha Lam Fanshawe DB 8134Kew1963Zambia Azima tetracantha Lam Bogdan A AB 4358Kew1956Kenya Azima tetracantha Lam Mogg AOD 28026Kew1958Mozambique Salvadora persica L Jansen PCMKew1981Mozambique Salvadora persica L Chase NO 6967Kew1958Zimbabwe Salavdora persica L Patel IH 833Kew1981Malawi Bataceae Batis maritime L Stork H E 8951Kew1943USA Batis maritima L Rehder A 827Kew1920USA Batis argillicola P Royen Wightman G 1816Kew1988Australia Koeberliniaceae Koeberlinia spinosa Zucc Badcock 489Kew1964Bolivia Koeberlinia spinosa Zucc Palmer 151Kew1880USA Emblingiaceae Emblingia calceolifl ora F Muell Oldfi eldKewc 1850Australia Tovariaceae Tovaria pendula Ruiz and PavonZak and Jaramilo 3216Kew1987Equador Pentadiplandraceae Pentadiplandra brazzeana Baill Breteler and De Wilde 670Kew1978Gabon Gyrostemonaceae Codonocarpus cotinifolius Desf F Muell Everist SL 7489Kew1963Australia Codonocarpus pyramidalis F Muell F Muell Greuter W 18736Kew1981Australia Gyrostemon australasicus Moq HeimerlMelville EJ 71 779Kew1971Australia Gyrostemon ramulosus Desf Lazarides and Palmer J 220Kew1988Australia Gyrostemon sheathii W Fitzg Maun V 120Kew1969Australia Gyrostemon spLazarides M 8294Kew1977Australia Tersonia brevipes Moq Bennett EM 2Kew1965Australia Resedaceae Ochradenus arabicus Chaudhary Hillc and A G Mill Thulin and Al Gifri 9984Kew1999United Arab Emirates Caylusea canescens St Hil Collenette 46Kew1977Saudi Arabia Oligomeris linifolia Vahl MacbrideBoulos and Cope 17700Kew1990Kuwait Capparaceae Atamisquea emarginata Miers ex Hook and Arn Kessler 4750WiscBolivia Atamisquea emarginata Miers ex Hook and Arn Hansen B 1388Wisc Belencita nemorosa Jacq Dugand Iltis 30550Wisc1991Venezuela Belencita nemorosa Jacq Dugand Iltis 30559Wisc1991Venezuela Boscia angustifolia A Rich Ash JKew1974Ethiopia Boscia angustifolia A Rich Nongonierma A 557Kew1967Senegal Boscia angustifolia A Rich Seyani JH and Balaka JL 1295Kew1983Malawi Boscia angustifolia A Rich Hepper FN and Wood JRI 5592Kew1975Yemen Boscia longifolia Hadj Moust Miller J 3799Wisc1988Madagascar Boscia senegalensis Pers Lam Fos